Wastewater sequencing reveals early cryptic SARS-CoV-2 variant transmission.

As SARS-CoV-2 continues to spread and evolve, detecting emerging variants early is critical for public health interventions. Inferring lineage prevalence by clinical testing is infeasible at scale, especially in areas with limited resources, participation, or testing and/or sequencing capacity, which can also introduce biases1-3. SARS-CoV-2 RNA concentration in wastewater successfully tracks regional infection dynamics and provides less biased abundance estimates than clinical testing4,5. Tracking virus genomic sequences in wastewater would improve community prevalence estimates and detect emerging variants. However, two factors limit wastewater-based genomic surveillance: low-quality sequence data and inability to estimate relative lineage abundance in mixed samples. Here we resolve these critical issues to perform a high-resolution, 295-day wastewater and clinical sequencing effort, in the controlled environment of a large university campus and the broader context of the surrounding county. We developed and deployed improved virus concentration protocols and deconvolution software that fully resolve multiple virus strains from wastewater. We detected emerging variants of concern up to 14 days earlier in wastewater samples, and identified multiple instances of virus spread not captured by clinical genomic surveillance. Our study provides a scalable solution for wastewater genomic surveillance that allows early detection of SARS-CoV-2 variants and identification of cryptic transmission.

Sentinel Cards Provide Practical SARS-CoV-2 Monitoring in School Settings.

A promising approach to help students safely return to in person learning is through the application of sentinel cards for accurate high resolution environmental monitoring of SARS-CoV-2 traces indoors. Because SARS-CoV-2 RNA can persist for up to a week on several indoor surface materials, there is a need for increased temporal resolution to determine whether consecutive surface positives arise from new infection events or continue to report past events. Cleaning sentinel cards after sampling would provide the needed resolution but might interfere with assay performance. We tested the effect of three cleaning solutions (BZK wipes, Wet Wipes, RNase Away) at three different viral loads: "high" (4 × 104 GE/mL), "medium" (1 × 104 GE/mL), and "low" (2.5 × 103 GE/mL). RNase Away, chosen as a positive control, was the most effective cleaning solution on all three viral loads. Wet Wipes were found to be more effective than BZK wipes in the medium viral load condition. The low viral load condition was easily reset with all three cleaning solutions. These findings will enable temporal SARS-CoV-2 monitoring in indoor environments where transmission risk of the virus is high and the need to avoid individual-level sampling for privacy or compliance reasons exists. IMPORTANCE Because SARS-CoV-2, the virus that causes COVID-19, persists on surfaces, testing swabs taken from surfaces is useful as a monitoring tool. This approach is especially valuable in school settings, where there are cost and privacy concerns that are eliminated by taking a single sample from a classroom. However, the virus persists for days to weeks on surface samples, so it is impossible to tell whether positive detection events on consecutive days are a persistent signal or new infectious cases and therefore whether the positive individuals have been successfully removed from the classroom. We compare several methods for cleaning "sentinel cards" to show that this approach can be used to identify new SARS-CoV-2 signals day to day. The results are important for determining how to monitor classrooms and other indoor environments for SARS-CoV-2 virus.

Implementation of Practical Surface SARS-CoV-2 Surveillance in School Settings.

Surface sampling for SARS-CoV-2 RNA detection has shown considerable promise to detect exposure of built environments to infected individuals shedding virus who would not otherwise be detected. Here, we compare two popular sampling media (VTM and SDS) and two popular workflows (Thermo and PerkinElmer) for implementation of a surface sampling program suitable for environmental monitoring in public schools. We find that the SDS/Thermo pipeline shows superior sensitivity and specificity, but that the VTM/PerkinElmer pipeline is still sufficient to support surface surveillance in any indoor setting with stable cohorts of occupants (e.g., schools, prisons, group homes, etc.) and may be used to leverage existing investments in infrastructure. IMPORTANCE The ongoing COVID-19 pandemic has claimed the lives of over 5 million people worldwide. Due to high density occupancy of indoor spaces for prolonged periods of time, schools are often of concern for transmission, leading to widespread school closings to combat pandemic spread when cases rise. Since pediatric clinical testing is expensive and difficult from a consent perspective, we have deployed surface sampling in SASEA (Safer at School Early Alert), which allows for detection of SARS-CoV-2 from surfaces within a classroom. In this previous work, we developed a high-throughput method which requires robotic automation and specific reagents that are often not available for public health laboratories such as the San Diego County Public Health Laboratory (SDPHL). Therefore, we benchmarked our method (Thermo pipeline) against SDPHL's (PerkinElmer) more widely used method for the detection and prediction of SARS-CoV-2 exposure. While our method shows superior sensitivity (false-negative rate of 9% versus 27% for SDPHL), the SDPHL pipeline is sufficient to support surface surveillance in indoor settings. These findings are important since they show that existing investments in infrastructure can be leveraged to slow the spread of SARS-CoV-2 not in just the classroom but also in prisons, nursing homes, and other high-risk, indoor settings.

SARS-CoV-2 Distribution in Residential Housing Suggests Contact Deposition and Correlates with Rothia sp.

Monitoring severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) on surfaces is emerging as an important tool for identifying past exposure to individuals shedding viral RNA. Our past work demonstrated that SARS-CoV-2 reverse transcription-quantitative PCR (RT-qPCR) signals from surfaces can identify when infected individuals have touched surfaces and when they have been present in hospital rooms or schools. However, the sensitivity and specificity of surface sampling as a method for detecting the presence of a SARS-CoV-2 positive individual, as well as guidance about where to sample, has not been established. To address these questions and to test whether our past observations linking SARS-CoV-2 abundance to Rothia sp. in hospitals also hold in a residential setting, we performed a detailed spatial sampling of three isolation housing units, assessing each sample for SARS-CoV-2 abundance by RT-qPCR, linking the results to 16S rRNA gene amplicon sequences (to assess the bacterial community at each location), and to the Cq value of the contemporaneous clinical test. Our results showed that the highest SARS-CoV-2 load in this setting is on touched surfaces, such as light switches and faucets, but a detectable signal was present in many untouched surfaces (e.g., floors) that may be more relevant in settings, such as schools where mask-wearing is enforced. As in past studies, the bacterial community predicts which samples are positive for SARS-CoV-2, with Rothia sp. showing a positive association. IMPORTANCE Surface sampling for detecting SARS-CoV-2, the virus that causes coronavirus disease 2019 (COVID-19), is increasingly being used to locate infected individuals. We tested which indoor surfaces had high versus low viral loads by collecting 381 samples from three residential units where infected individuals resided, and interpreted the results in terms of whether SARS-CoV-2 was likely transmitted directly (e.g., touching a light switch) or indirectly (e.g., by droplets or aerosols settling). We found the highest loads where the subject touched the surface directly, although enough virus was detected on indirectly contacted surfaces to make such locations useful for sampling (e.g., in schools, where students did not touch the light switches and also wore masks such that they had no opportunity to touch their face and then the object). We also documented links between the bacteria present in a sample and the SARS-CoV-2 virus, consistent with earlier studies.

Analysis of SARS-CoV-2 RNA Persistence across Indoor Surface Materials Reveals Best Practices for Environmental Monitoring Programs.

Environmental monitoring in public spaces can be used to identify surfaces contaminated by persons with coronavirus disease 2019 (COVID-19) and inform appropriate infection mitigation responses. Research groups have reported detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) on surfaces days or weeks after the virus has been deposited, making it difficult to estimate when an infected individual may have shed virus onto a SARS-CoV-2-positive surface, which in turn complicates the process of establishing effective quarantine measures. In this study, we determined that reverse transcription-quantitative PCR (RT-qPCR) detection of viral RNA from heat-inactivated particles experiences minimal decay over 7 days of monitoring on eight out of nine surfaces tested. The properties of the studied surfaces result in RT-qPCR signatures that can be segregated into two material categories, rough and smooth, where smooth surfaces have a lower limit of detection. RT-qPCR signal intensity (average quantification cycle [Cq]) can be correlated with surface viral load using only one linear regression model per material category. The same experiment was performed with untreated viral particles on one surface from each category, with essentially identical results. The stability of RT-qPCR viral signal demonstrates the need to clean monitored surfaces after sampling to establish temporal resolution. Additionally, these findings can be used to minimize the number of materials and time points tested and allow for the use of heat-inactivated viral particles when optimizing environmental monitoring methods. IMPORTANCE Environmental monitoring is an important tool for public health surveillance, particularly in settings with low rates of diagnostic testing. Time between sampling public environments, such as hospitals or schools, and notifying stakeholders of the results should be minimal, allowing decisions to be made toward containing outbreaks of coronavirus disease 2019 (COVID-19). The Safer At School Early Alert program (SASEA) (https://saseasystem.org/), a large-scale environmental monitoring effort in elementary school and child care settings, has processed >13,000 surface samples for SARS-CoV-2, detecting viral signals from 574 samples. However, consecutive detection events necessitated the present study to establish appropriate response practices around persistent viral signals on classroom surfaces. Other research groups and clinical labs developing environmental monitoring methods may need to establish their own correlation between RT-qPCR results and viral load, but this work provides evidence justifying simplified experimental designs, like reduced testing materials and the use of heat-inactivated viral particles.